This invention relates to assays for evaluating tumor factors which affect breast cancer. The assays for Ubc9 binding are particularly useful for evaluation of tumor activity or expected activity. The methods of the invention are also useful for identifying tumorogenic agents. The methods of the invention are also useful for identification of mutations of a naturally occurring short form of BRCA1 protein named BRCA1a which is expressed at reduced levels or undetectable in high grade breast and ovarian cancers.
BRCA1 and BRCA1a are two proteins that differ in the size and are products of the BRCA1 gene. BRCA1 (breast cancer 1) is a human gene, some mutations of which are associated with a significant increase in the risk of breast cancer (early onset), as well as other cancers. BRCA1 belongs to a class of genes known as tumor suppressors, which maintains genomic integrity to prevent uncontrolled proliferation.
Inherited mutations of the breast cancer susceptibility gene 1 (BRCA1) confer a high risk for the development of estrogen (E2) dependant breast and ovarian cancers. The underlying basis for its tissue specific tumor suppressor function remains poorly understood. Multiple naturally occurring isoforms of BRCA1 are present in different tissues with varying expression levels that encode proteins missing all or part of exon 11. BRCA1a/p110 and BRCA1b/p100 form two of the four major splice variants present in normal, breast and ovarian cancer cells. Both BRCA1a and BRCA1b differ from BRCA1 in having a deletion of the majority of exon 11 sequences (within amino acids (aa) 263-1365). BRCA1b has an additional deletion of exons 9 and 10 sequences and they code for 110 kDa/100 kDa proteins (FIG. 1). BRCA1 and its splice variants are multifunctional proteins that interact with several proteins which regulate a number of biological activities like transcriptional activation/repression, cell-cycle regulation, growth/tumor suppression, apoptosis, DNA repair, genomic stability, steroid hormone receptor signaling, ubiquitination and sumoylation but many of these functions are not tissue specific. Women carrying BRCA1 mutations develop breast tumors that are triple negative (TN) suggesting that hormonal factors play a critical role in the development of these cancers. TN breast cancers account for only about 15% of all breast cancers, they represent a higher percentage of breast cancers arising in premenopausal women and in African American women. currently there are no targeted therapies available for these cancers (14). There is significant overlap between TN breast cancers and BRCA1 associated breast cancers both histologically as well as transcriptionally, which suggests that dysfunction in the BRCA1 pathway may be responsible for the development of these cancers. Estrogen receptor (ERα) signaling has been implicated in the development of BRCA1 associated tissue specific tumorogenesis.
The majority of BRCA1-related breast cancers are ER-negative and more prevalent in younger African American women and pre-menopausal women with breast cancers. (Tischkowitz M D, Foulkes W D: The basal phenotype of BRCA1-related breast cancer: past, present and future. Cell Cycle 5: 963-7, 2006.). Based on recent findings it is shown that lack of BRCA1 results in ER-negative tumors due to sumoylation of ERα resulting in transcriptional silencing of ERα expression. BRCA1 proteins may be an integral part of the sumoylation machinery that function to regulate the overall sumoylation activity of ERα activity in breast and ovarian cancer cells similar to the RNF4 family of RING-finger E3 ubiquitin ligase (Sun H, Leverson J, Hunter T: Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins. EMBO 26: 4102-12, 2007.). We can also speculate that BRCA1 belongs to the family of SIM-containing RING-finger proteins. (Uzunova K, Gottsche K, Miteva M, Weisshaar S, Glanemann C, Schnellhardt M, Niessen M, et al.: Ubiquitin-dependent Proteolytic Control of SUMO Conjugates. J Biol Chem 282: 34167-75, 2007) that induce turnover of ERα following its sumoylation. The role of BRCA1 may be to fine tune ERα transcriptional activation in response to rapid changes in E2 levels. Assays such as that shown in FIG. 1 that measure the extent to which BRCA1 dysfunction results in ERα-positive and negative breast cancers are needed.
FIG. 1 gives models showing how BRCA1 dysfunction in regulating the E2-induced ER α-activation/repression by SUMO-dependent/independent activities of Ubc9 results in (a) ERα-negative and (b) ERα-positive breast cancers.
Post translational modification of transcriptional factors is important for regulated gene expression. SUMO-1, a 98 amino acid polypeptide, is covalently attached to lysine residues in proteins. Post-translational modification by SUMO has effects on the stability, localization, protein-protein interactions and transcriptional regulation (activation or repression). In most cases sumoylation of transcription factors (eg. HDAC1, p300/CBP, CtBP, STAT1, etc.) and Histone 4 inhibits transcription by promoting recruitment of co-repressors like HDAC complex. SUMO-1 also binds to the SUMO binding motifs in BRCA1 and represses BRCA1-mediated transcription in a sumo-independent manner by recruiting HDAC. The SBM is different from the SUMO-1 modification consensus sequence (Ψ-K-X-E) which is found in SUMO-1 substrates proteins (Table 1 and FIG. 1a/b). SBM binds SUMO non-covalently but Ψ-K-X-E does not bind to SUMO-1 non-covalently. It appears to bind Ubc9 non-covalently for covalent SUMO attachment. Most SUMO-modified proteins contain the tetra peptide motif Ψ-K-X-D/E where Ψ is a hydrophobic residue, K is the lysine conjugated to SUMO, X is any amino acid, and D or E is an acidic residue. A wide variety of proteins are sumoylated (Table 1). ERα is a nuclear transcription factor that undergoes various types of post translational modifications like phosphorylation, acetylation, ubiquitination, methylation, and sumoylation. ERα is sumoylated at conserved lysine residues within the hinge region only in the presence of E2. PIASI, PIAS3 act as E3 ligases and Ubc9 as E2 SUMO-conjugating enzyme for ERα sumoylation and also modulate ERα transcription independent of SUMO-1 conjugation activity. Mutations that abrogate sumoylation impair ERα dependent transcriptional activation but not its subcellular localization. Ubc9 is the only mammalian E2 conjugating enzyme that is essential for sumoylation. The mutant C93S Ubc9, which prevents SUMO-1 conjugation by preventing the formation of thiolester bond between SUMO-1 and Ubc9, still functions as a co activator for nuclear receptors. Thus there can be sumo-dependent transcriptional activation or repression and sumo-independent transcriptional activation or repression of promoters. A role for post translational modifications in targeting ERα for degradation is poorly understood. A direct correlation has been observed by some between the rate of ERα degradation and its transcriptional activation and treatment of cells with proteosome inhibitor MG132, impaired ERα transcription. Furthermore ERα is ubiquitylated after the first round of transcription which may be needed for subsequent E2-mediated ERα transcription. ERα thus cycles on and off the promoter as long as E2 is present. SUMO-1 was found to suppress BRCA1 mediated transcription of Gadd45 α, p27 KIP1 and p21 WAF1/CIP1 via modulation of promoter occupancy. The only known enzymatic activity that is associated with BRCA1 is its E3 ubiquitin ligase activity, and recently ERα was shown to be a putative substrate for this BRCA1/BARD1 ubiquitin ligase activity. Deleterious BRCA1 RING-finger domain mutations eliminated the ubiquitin ligase activity, but did not eliminate its auto ubiquitination activity indicating a link between tumor suppressor function of BRCA1 and its E3 ligase activity. However all these studies do not uncover the paradox as to why impairment of this E3-ligase activity contributes to ER-negative breast cancers.
Several patents have been issued relating to the BRCA1 gene and protein. U.S. Pat. No. 5,750,400 to Murphy, et al., which is incorporated herein in its entirety, discloses and claims the gene sequences. It also discloses means of identifying certain mutations. U.S. Pat. No. 5,747,282 to Skolnick, et al, which is incorporated herein by reference in its entirety, discloses 17q-linked sequence of a gene predisposing to breast and ovarian cancer. U.S. Pat. No. 5,756,294 to White, et al, which is incorporated herein by reference in its entirety, discloses another cDNA sequence which correlates with susceptibility to breast and ovarian cancer and uses allele-specific oligonucleotides in identifying the particular sequences. U.S. Pat. No. 7,507,800 to van Ommen, et al, which is incorporated by reference herein in its entirety, discloses and claims a diagnostic kit for determining predisposition to breast and ovarian cancer. The test relies on use of probes for particular, previously identified sequences.